Epidermal growth factor (EGF) is a 53-amino acid protein synthesized in the duodenum and salivary glands of normal humans, and normally excreted in the urine. For its effect in reducing gastric acid secretion, and its first isolation source, it was formerly termed urogastrone. After it was sequenced, it was recognized that urogastrone was homologous to murine EGF, and that urogastrone additionally stimulated the proliferation of certain cell types, prompting a change in nomenclature to EGF. The biological and chemical properties of hEGF and mEGF are reviewed in G. Carpenter et al, Ann Rev Biochem (1979) 48:193-216.
The amino acid sequence of human EGF is:
Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg (SEQ ID NO:1)
This sequence was published by H. Gregory et al, Int J Peptide Protein Res (1977) 9:107-18, who isolated the protein as urogastrone from human urine (1 mg/ 1000 L), and disclosed its sequence homology with mEGF. Murine EGF has the sequence (differences from hEGF underlined):
Asn Ser Tyr Pro Gly Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu Asn Gly Gly Val Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys Asn Cys Val Ile Gly Tyr Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu Arg Trp Trp Glu Leu Arg, (SEQ ID NO:2)
which conserves the disulfide bonds. Gregory also discloses that urogastrone may be maintained in aqueous solution at pHs of 1-11 for at least 20 hours without loss of activity, and that the six C-terminal amino acids may be removed without loss of biological activity.
Preparation of an hEGF gene is described in EPO 046,039, and cloning and expression of EGF is disclosed in commonly-owned copending U.S. patent application Ser. No. 004,212, filed Jan. 5, 1987, now U.S. Pat. No. 5,096,825, a continuation of Ser. No. 457,412, filed Jan. 12, 1983, now abandoned. U.S. Ser. No. 004,212 now U.S. Pat. No. 5,096,825, is incorporated herein by reference in full. Other disclosures of EGF preparation are M. S. Urdea et al, Proc Nat Acad Sci USA, (1983) 80:7461-65 (chemical synthesis of gene and expression in yeast), T. Oka et al, Proc Nat Acad Sci USA, (1985) 82:7212-16 (fusion protein in E. coli); Cohen et al, U.S. Pat. No. 4,743,679 (recombinant fusion protein); Sugimoto, U.S. Pat. No. 4,621,052 (human hybridoma cell culture); Nishimura et al, U.S. Pat. No. 4,528,186 (adsorption from urine); and Cohen et al, U.S. Pat. No. 3,948,875 (purification from murine submaxillary glands). Pharmaceutical compositions containing EGF are disclosed in Finkenaur, U.S. Pat. No. 4,717,717 (stabilized against degradation by moisture with water-soluble cellulose derivatives); U.S. Pat. No. 4,703,108 (cross-linked collagen sponge); and Camble et al, U.S. Pat. No. 3,917,824 (lyophilized solid or dextrose solution).
C. George-Nascimento et al, Biochem (1988) 27:797-802 reported the isolation from recombinant yeast culture of four distinct forms of EGF, termed A, B, C, and D, each of which exhibit full EGF activity. EGF-D represents the 52-amino acid sequence obtained by removing the C-terminal arg, while EGF-B corresponds to EGF-D wherein the C-terminal arg-leu has been removed. EGF-C appears to be EGF-D in which Met.sub.21 has been oxidized, while EGF-A appears to be EGF-B with Met.sub.21 oxidized. EGF-D is reported to be stable when stored as a lyophilized powder.
George-Nascimento et al, U.S. Ser. No. 07/351,773, now U.S. Pat. No. 5,158,935, disclosed EGF muteins in which the Asp.sub.11 residue is replaced, preferably with Glu, in order to prevent degradation by isomerization of the Asp residue. Asp.sub.11 may isomerize to iso-Asp, which disturbs the peptide backbone and impairs the peptide's activity.
The physical structure of a recombinantly-produced hEGF has been partially elucidated using COSY and NOESY by K. Makino et al, Proc Nat Acad Sci USA (1987) 84:7841-45. Makino disclosed that amino acids 19-32 form an antiparallel beta-pleated sheet, placing His.sub.10 in close proximity to Tyr.sub.22 and Tyr.sub.29. Makino also suggested that amino acids 45-53 may form an alpha helix which crosses the surface of the beta-pleated sheet, creating a hydrophobic pocket comprising His.sub.10, Tyr.sub.22, Tyr.sub.29, Trp.sub.49, and Trp.sub.50. Removal of amino acids 49-53 altered the NMR chemical shift and pKa of the ring protons on His.sub.10, His.sub.19, Tyr.sub.22, and Tyr.sub.29, and drastically reduced the activity, suggesting that these amino acids may participate in the EGF binding site.
Nestor et al, U.S. Pat. No. 4,686,283 disclosed the preparation of polypeptides and polypeptide analogs homologous to amino acids 34-43 of EGF and TGF-.alpha., which are useful as EGF antagonists and for preparing anti-EGF antibodies.